Artificial blood vessels and method of preparing the same



Feb. 4, 1969 M. CHVAPIL ET AL 3,425,413

ARTIFICIAL BLOOD VESSELS AND METHOD OF PREPARING THE SAME Filed April15, 1964 United States Patent 1962, 5,644/62 U.S. Cl. 128-334 5 ClaimsInt. Cl. A6111 17/04; A61f l /24; A61] 17/00 ABSTRACT OF THE DISCLOSUREAn artificial blood vessel includes a tanned collagen tube strong enoughto be self-supporting, and impregnated with an anti-coagulant which iscovered with open mesh fabric secured to the tanned collagen by an outerwall portion of collagen in the native chemical state thin enough not toclose the pores in the fabric.

This application is a continuation-in-part of our copending applicationSer. No. 330,221, filed on Dec. 10, 1963, and now abandoned.

This invention relates to vascular grafts, that is, to artificial bloodvessels, and to a method of preparing the same.

As described in more detail in a paper published by us and on our behalfin the Journal of Surgical Research (vol. 3, No. 7, pages 358 to 368,Sept. 1963), a vascular graft should be non-toxic, flexible, and porous.It should retain its strength permanently in intimate contact with bodyfluids, and should be readily accepted and incorporated into thesurrounding tissue. The graft or prosthesis, moreover, should notunfavorably affect the flow of blood therethrough.

In the surgical repair of blood vessels and body ducts, inert fabricshave found considerable use. The known inert fabric prostheses, however,do not become parts of the body tissues. They frequently remainsurrounded by a pool of serum after the healing process is terminated.We have found that the relatively low porosity of the known prosthesesaccounts for the observed formation of fluid pockets, and prevents thegrowth of repair tissue through the fabric grafts.

The porosity of a fabric may be characterized by the volume of Water inmilliliters per minute that will pass through one square centimeter ofthe fabric under a pressure differential of 120 millimeters mercury andthis definition of porosity is being employed in this specification andthe appended claims. The vascular fabric prostheses that have found usein surgery heretofore have a porosity of less than 2,000 milliliters perminute.

It has now been discovered that healing will be greatly improved and thegrowth of repair tissue through the fabric promoted if the porosity ofthe fabric is much greater, that is, in the range of 10,000 to about22,000 milliliters per minute. A vascular graft of the invention thusincludes a fabric of the desired porosity. Such a fabric is not initself impervious to blood.

In the improved prostheses of the present invention, the porous,non-absorbable fabric framework is rendered bloodtight by a collagenfilm or tube bonded to one side of the fabric. The fabric is exposed tothe surrounding tissue thus permitting ingrowth of fibroblasts andendothelial cells which results in the attachment of the prosthesis tothe host tisues. The collagen has considera- 3,425,418 Patented Feb. 4,1969 ble tensile strength, is non-antigenic, and is slowly absorbed at arate which may be controlled by partial tanning so that there is noappreciable decrease in the strength of the vessel structure while thecollagen is being replaced by new body tissue.

It has further been found that clotting of blood in vascular grafts ofeven very small internal diameter (three millimeters and less) can beprevented if the interior collagen surface of a vascular prosthesis ofthe invention contains an anti-coagulant such as heparin. Theanticoagulant properties of natural blood vessels are ascribed to thepresence of mucopolysaccharides, particularly chondroitin sulfate B andheparitin sulfate. We may therefore add these or other anticoagulants tothe collagen structure as a whole, or we incorporate an anticoagulantonly in the inner wall portion of the collagen tube to decrease thethrombotic effects of the graft. We have found that heparin is quicklyadsorbed from aqueous solutions by a partly tanned collagen wall, andcannot readily be removed from the wall under ordinary physiologicalconditions. A collagen wall containing heparin has permanentanticoagulant properties.

While the present invention is not limited to any particular theory ofaction, it is believed that heparin and other anticoagulants arereversibly bound to the collagen through polar groups present in theanticoagulants and in the collagen, and that the anticoagulant is slowlyreleased into the bloodstream over a period of days as the collagen isabsorbed by the body. Thus, the probability of clotting is sharplyreduced during the first few days after the implantation of the graftwhen the patient is most susceptible to this danger, and an open lumenis maintained during the deposition and organization of the fibrinlining surrounding the graft.

Added mucopolysaccharides also increase the hydropliilic properties ofthe collagen, and they improve the physicochemical stability of thecollagen by the presumed link between the mucopolysaccharides and thecollagen molecules. The hydrophilic properties of the absorbable tubeare also greatly enhanced by the admixture of glycerin which ensureselasticity of the tube even after tanning.

The artificial blood vessels of the inveniton may be sterilized byradiation or by chemical means in a known manner. They may be storedover extended periods without loss of their desirable properties. Smallamounts of antibiotics added to the collagen during the preparation ofthe vessel and contained in the adsorbable walls thereof further ensuresafe storage and have the expected beneficial results afterimplantation.

An outer wall portion of the collagen layer adjacent the fabric layer ispreferably kept untanned, that is, in the native chemical state andamorphous. The untanned outer wall portion is adhered to the fabriclayer, but does not obstruct access from the outside to the pores of thefabric.

Fabrics well suited for the vascular grafts of the invention includethose made from polyester fibers such as polyethylene terephthalatewhich are sold under the registered trademarks Dacron and Terylene.Polytetrafluoroethylene fibers, such as those 'known under the trademarkTeflon also may be employed, but are less resilient and are not soconveniently fabricated as the polyester fabrics. Thepolytetrafluoroethylene fabrics, however, have superior healingproperties and produce thinner, more compact capsules.

The non-absorbable fabric that reinforces the collagen may be preparedin tubular form by braiding, weaving, or knitting. Braided grafts lackdimensional stability and are not widely used, whereas, both woven andknitted grafts have been used clinically. Woven grafts must be tightlyconstructed or fraying and tfiber slippage become problems atimplantation. -It is for this reason that woven grafts havecomparatively low porosities. Knitting permits variations in thetightness or porosity without commensurate penalties in fray or slipqualities, and is the preferred method of construction.

Other features and many of the attendant advantages of this inventionwill be readily appreciated as the same becomes better understood byreference to the following detailed description when considered inconnection with the accompanying drawing in which:

FIG. 1 is a perspective view of an extruded collagen tube;

FIG. 2 is a perspective view of a non-absorbable fabric tube;

FIG. 3 is a greatly enlarged view of the nonabsorbable knitted fabricthat forms the reinforcing structure illustrated by FIG. 2;

FIG. 4 is a greatly enlarged view of a section of a vascular prosthesisconstructed in accordance with the present invention;

FIG. 5 is a fragment of FIG. 4, enlarged; and

FIG. 6 is a view along the line 6-6 of FIG. 5.

Referring now to FIGS. 1, 2, and 4, a vascular graft may be constructedin accordance with the present invention by slipping a collagen tubeinside of a fabric tube. The collagen tube is softened by soaking inwater or a dilute aqueous acid solution and is then expanded against theexternal fabric tube. A convenient way to accomplish this is to increasethe pressure of the air within the tube, and thereby force the collageninto contact with the inner wall of the fabric. In so doing the collagenis forced against the fabric and into the interstices between thenon-absorbable fibers. The structure is then air-dried whereby the innercollagen tube is stabilized in the form illustrated in FIGS. 4, 5, and6.

The non-absorbable fabrics may be knitted, crocheted, woven, or braidedin the shape of the desired prosthesis, as a film, tube, Y-tube, etc.While the drawings illustrate a tubular structure suitable for use as avascular graft, it will be understood that other shapes in whichcollagen is bonded to one side of a non-absorbable fabric characterizedby a porosity not less than 10,000 milliliters per minute and notsubstantially more than 22,000 milliliters per minute offer similaradvantages.

It will be understood that the collagen present in the prostheses of thepresent invention may be treated with tanning agents such asformaldehyde, pyrogallol, chromium, etc., by methods well known in theart to obtain increased strength, and to control the rate at which thecollagen will be absorbed.

A combination fabric-collagen vascular graft may also be manufactured byconstructing the tubular fabric framework of Dacron or Teflon to thedesired diameter. The fabric framework is then coated on both sides witha collagen mass obtained by swelling collagen fibrils in an aqueous acidsolution. The swollen collagen fibrils are then frozen in position anddeswollen by dehydration in an organic solvent. Finally the collagenthat lines the interior wall of the composite tube is treated withheparin.

In this type of construction, it is important that the mesh of thenon-absorbable fabric be sufficiently open to permit the collagenfibrils to extend into and through the interstices of the fabric. Thesecollagen fibrils that pass through the fabric cohere to the collagenfibrils on the other side of the fabric framework and form a unitarystructure that resists delamination. It is preferred that only theinterior wall of the graft be treated with heparin as the ingrowth offibroblasts and endothelial cells appears retarded by the presence ofheparin on the exterior surface of the graft.

The improved prostheses of the present invention may also be constructedby weaving, knitting, Crocheting, or braiding together an inertnon-absorbable thread or yarn with collagen yarn or collagenmultifilament. Again the interior surface of this tubular structure istreated with heparin.

In applying heparin t0 the interior collagen wall an aqueous solutioncontaining from 0.25% to 1% heparin may be used. The contact time ofthis solution with the collagen should be sufiicient to permit theheparin to penetrate about 70% of the distance through the wall of thetube. For the reasons indicated above it is preferred that there be noheparin on the exterior wall of the tube.

The present invention is more fully described and exemplified in thefollowing examples. It is to be understood, however, that our inventionis not to be limited to any specific form of materials or conditions setforth in the examples, but is limited solely by the appended claims.Throughout the examples which follow, all quantities are expressed inparts by weight.

Example I A rigid, self-supporting collagen tube having an internaldiameter of about 10 millimeters and a wall thickness of about 1millimeter was extruded by the procedure described in the Becker PatentNo. 2,246,236, using a plastic collagen mass containing about 30%collagen by weight. The extruding mechanism was that described in theBecker Patent No. 2,115,607 and resulted in orientation of the collagenfibers to give excellent strength characteristics.

The tube so obtained was placed within a woven Dacron tube having aporosity of 22,000 milliliters per minute and the assembly was immersedin water until the collagen became plastic. One end of the prosthesiswas then closed and sufiicient air pressure was applied to the other endof the prosthesis to force the collagen against the inner wall of thefabric. The fabric-collagen tube was then airdried in an oven at 45 C.overnight without damage. The product so obtained is illustrated inFIGS. 4, 5, and 6.

One end of the collagen-Dacron prosthesis was again closed and the tubewas filled with an aqueous solution containing 1.0% of heparin. Theheparin used, standardized by the sheep plasma coagulation method, wasfound to contain 100 units of anticoagulant activity per milligram ofdry material. After the heparin solution had penetrated aboutthree-fourths of the distance through the wall of the collagen tube, theprosthesis was emptied of the heparin solution and air-dried overnight.

The collagen-Dacron prosthesis was next tanned by immersing it for 30seconds in a solution of 0.4 part of pyrogallol, 0.1 part .tetrasodiumethylenediamine tetraacetic acid, and 99.5 parts of water adjusted to pH8.3 with ammonium hydroxide. It was then re-dried in an oven at 50 C.for 6 hours.

The prosthesis was next immersed for 30 seconds in a solution ofchromium (III) sulfate comprising 0.8 part of chromium as chromic oxide,0.5 part of lactic acid 0.24 part of formaldehyde, and 98.46 parts ofwater, adjusted to pH 2.7 with sodium hydroxide. The prosthesis wasagain dried in an oven at 50 C. overnight. The finished prosthesis wassterilized by irradiation.

Example II Fresh steer hides were washed with cold water at 13 C. orless in a rotating drum for 10 to 24 hours. After washing, the hideswere defleshed with a scraping machine, and the hair and epidermis werecut off with a horizontal band knife. This preliminary cleaning wasaccomplished with standard tannery equipment.

The remaining hair and poorly cleaned sections were cut off by hand andcompositions were prepared from five hides. The hide composites werethen cut into /2 to 4 square inch sections and reduced to pulp by threepasses through a meat grinder, each pass being a finer grind. The firstand second passes were through 18 and 8 millimeter holes, respectively.The final grind was through holes 1.5 millimeters in diameter. Specialcare was taken during the grinding process to keep the pulp below C.This was done by adding crushed ice to the hide sections as they werefed to the grinder.

The ground pulp was next diluted with tap waterat 16 C. to give a smoothslurry containing dry solids. To this slurry was then added lactic acid,and the mixture was kneaded to form a homogeneous mass of swollencollagen fibers. The mixture so obtained contained about 25% by weighthide solids.

.The collagen mass was extruded as described in Example I to form acollagen tube having a diameter of about 8 millimeters and a wallthickness of about 0.4 millimeter.

This extruded tube was placed within a Teflon fabric tube of the typeillustrated in FIG. 2, characterized by a porosity of 10,000milliliters, and expanded against the interior wall of the fabric asdescribed in Example I.

The vascular prosthesis so formed was tanned by immersing it in anaqueous solution containing 2% trimethylolmelamine (Solarpret), anddried in an oven at 45 C. overnight. It was further treated with heparinas in Example 1.

Example III The deep flexor tendon of cattle was cleaned of fat,superficial non-collagenous protein and other extraneous. matter and wassliced on an electric meat-slicing rna'chine (rotary knife) in thefrozen condition. The tendon sections were sliced perpendicularly totheir longitudinal axis to a thickness of about 11 mils. An aliquotsample of the tendon slices Was analyzed; the dry solids amounted to36.97%.

The sliced tendon was next treated with an enzyme solution to dissolveelastin. This enzyme solution was prepared by dissolving 0.15 part officin and 3.75 parts of ethylene diamine tetrasodium tetraacetate in 750parts of water. Seventy-five parts of the sliced tendon was immersed inthis solution which was stored at room temperature overnight. Then 2.25parts of hydrogen peroxide were added to destroy any residual ficin.

To this mixture of tendon slices in about 750 parts of water were addedan additional 2244 parts of water and 5.87 parts of cyanoacetic acid.The swelling solution was cooled to below 25 C. This mixture was stirredin a dis.- persion kettle at about 60 rpm. The remaining steps in theprocess were carried out at a temperature below about 25 C. and thetemperature of the collagen dispersion was not allowed to exceed thistemperature.

Stirring was continued for about 3 hours, during which time theindividual collagen slices were swollen. The dispersion was homogenizedby repeated passes through series-connected jets having orifices of 50mils and mils respectively. The dispersion was then forced through aleaf filter containing three screens of #316 stainless steel. Thesescreens were separated by A;-inch spacers and decreased in mesh size sothat the dispersion first passed a l44mil screen, then a 9-mil screen,and finally a 4-mil screen. The dispersion of swollen solvated collagenfibrils so obtained analyzed 1.09% solids and had a pH of 2.52.

A glass tube having an inside diameter of about A- inch was fitted witha one-hole stopper of rubber through which a i -inch :glas rod wasplaced so that the glass rod extended coaxially within the glass tube.Before placing the glass rod and rubber stopper in position, the glassrod was covered with a piece of rubber tubing, and a cylindrical tube ofopen-mesh woven Dacron about inch in diameter was slipped over the glassrod and rubber tube. The glass tube and glass rod were assembled in anupright position with the bottom of the Dacron fabric tube resting onthe rubber stopper. The dispersion of swollen collagen fibrils, preparedas described above, was poured into the glass tube while maintaining thefabric tube in a coaxial position and equally spaced between the rubbertube and glass tube so that both sides of the fabric were coated withthe collagen dispersion. This mold with the dispersion and fabric inplace was frozen in the vertical position for at least 4 hours at -20 C.

'The mold was then placed in a static coagulation bath consisting of 2liters of isopropanol, 30 cubic centimeters of concentrated ammonia (25and 10 cubic centimeters of formaldehyde (37% solution) at roomtemperature, and the mold was maintained in the solution for 16 hours.The glass rod covered with the rubber tube and the formed collagen tubewere then removed and placed in a dehydrating bath consisting of 2liters of isoproponal. The collagen tube was left in this bath for anadditional 16 hours to complete the dehydration.

After dehydration, the rubber tube with the collagen tube on it was verycarefully slid from the glass rod, and the rubber tube was removed fromthe interior of the collagen tube by pulling on both ends of the rubbertube, thereby stretching the rubber tube and reducing its diameter.After the collagen tube was removed from the rubber tubing, it wasplasticized in a bath consisting of 2 liters of isopropanol (10% water)containing 5% glycerine. This plasticizing operation is optional. After24 hours in the plasticizing bath, the collagen tube was supported on aglass rod and air-dried.

One end of the collagen-Dacron prosthesis so obtained was closed and thetube was filled with an aqueous solution containing 0.25% heparin. Whenthe heparin solution had penetrated three-fourths of the way through thecollagen-Dacron tube, the tube was emptied and airdried.

Example IV Hide sections of beef cattle were comrn'inuted andhomogenized together with a dilute solution of sodium hydroxide for 24hours, 60 milliliters 1/100 N NaOH being added per grams of the hidematerial. The homogenized mass was left standing in a refrigerator for24 hours at a temperature between 0 and 4 C. whereupon it congealed intoa soft gel.

During homogenizing, there were added 0.5 percent hyaluronic acid (amucopolysaccharide), 3 percent glycerin, and 1 gram chlorotetracyclinper 100 grams of hide material.

The gel was extruded in the manner described in Example I to form a tubeof 9 millimeter external diameter and a wall thickness of 0.5millimeter. Air was simultaneously blown into the axial cavity of theextruded tube at a pressure which was controlled between 40 and 150 mm.water column to prevent collapse of the extrudate.

The extruded tube was dried to constant temperature at ordinary roomtemperature (15 to 25 C.) in a gentle stream of air. A very thin layerof amorphous unmodified collagen was applied to the dry outer surface byspraying from a gun, and a loosely knit tube of smooth, noncrimpedTerylene was slipped over the tube in contact with the freshly depositedcollagen layer.

The fabric, of conventional weft knit construction, had a permeabilityto water of 18,000 ml./cm. /min. at mm. Hg. It was elasticallystretchable about 15% longitudinally as well as transversely. While someof its threads were adhered to the collagen tube by the freshlydeposited collagen layer, the porosity of the fabric was virtuallyunatfected by its engagement with the resorbable material.

The tube was filled with an aqueous 1% solution of2,4,6-trimethoxytriazine, a commercial tanning agent commonly sold underthe name Solapret. The tanning solution was removed after ten minutes,and the tube was washed out with water to remove residual unreactedtanning agent. The treatment was then repeated with aqueous 1% heparinsolution which was left in contact with the inner wall of the tube forfive minutes. The hepar'in penetrated about A of the collagen wall. Thetube was emptied and air-dried overnight.

The completed artificial blood vessel was sealed in a glass ampoule andwas sterilized by exposure over five hours to gamma radiation of 2x10roentgen.

The method outlined above may be modified in many respects withoutdeparting from the spirit and scope of this invention. It is notnecessary fully to dry the collagen tube prior to covering the same withfabric. Actually, it is possible to encase a freshly extruded wetcollagen tube in a tubular fabric envelope, and to adhere the fabric tothe collagenous material by expanding the tube under internal airpressure. The pressure has to be carefully matched to the mechanicalstrength of the collagen tube to avoid embedding the fabric material incollagen to an extent which would make the pores of the fabricunavailable or not readily available. The use of a sprayed or brushedouter coating of unmodified collagen may be dispensed with in such acase, but it may also be omitted where a fabric layer is applied todried collagen, and high mechanical strength of the vascular graft isnot required.

The composition of the collagen tube may be varied as to the minoradmixtures. Hyaluronie acid thus may be replaced by othermucopolysaccharides or mixtures thereof, such as those derived in aconventional manner from the corpus vitreum or cartilage of beef cattle.The mucopolysaccharides have distinctly noticeable effects on thestability of the gel when present in amounts of as little as 0.5percent. We have not observed further desirable changes when more than 3percent of the mucopolysaccharides were added to those naturally presentin the collagen material after processing.

Glycerin is beneficial in amounts of at least 0.5 percent, and may beincreased to five percent with corresponding improvement of elasticityand plasticity of the collagenous material, even when partially tanned.

The effects and dosages of antibiotics are well known from theliterature on the use of implants of a similar nature. Merely by Way ofexample, it may be stated that suitable antibiotics includechlortetracycline in amounts of 1 to 5 grams per 100 grams of thecollagen mass. 0.5 to 5 grams neomycin, or 10,000 to 50,000 unitsbacitracin. The quantitative limits indicated are not critical nor isthis list complete.

While it is critically important that the resorption and swelling ratesof the collagen be reduced by hardening, the hardening reagent and thehardening method may be freely chosen from the tanners art. Synthetictanning agents are preferred because of the very close reproducibilityof their results, but we have successfully employed natural tanningextracts of Chinese gallnu'ts, oak, pine, quebracho, sumach, mimosa, andvalonia.

Many other modifications and variations of the present invention arepossible in the light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

This application is a continuation-in-part of our copending applicationSer. No. 330,221 filed on Dec. 10, 1963.

We claim:

1. An artificial blood vessel of tubular laminar structure comprising,in combination:

(a) an exposed outer tubular layer of a physiologically inert fabricresistant to resorption by body fluids,

(1) said outer layer having a permeability to water of not less than10,000 milliliters per square centimeter per minute at a pressuredifferential of millimeters mercury column; and

(b) a tubular composite layer essentially consisting of two laminae ofcollagen,

(1) one of said laminae of collagen being tanned and constituting aninner and self-supporting Wall of said vessel,

(2) said inner wall containing an amount of an anti-coagulant effectiveto prevent coagulation of blood in contact with said inner wall, and

(3) the other of said collagen laminae being essentially in the nativechemical state and being bonded to said outer layer, said outer layerand said other lamina constituting an outer wall of said vessel.

2. A vessel as set forth in claim 1, wherein said other lamina ofcollagen is of a thickness sufiicient to adhere said fabric to saidinner wall Without closing the pores of the fabric.

3. A vessel as set forth in claim 1, wherein said anticoagulant includesa mucopolysaccharide admixed to said collagen.

4. A vessel as set forth in claim 1, wherein said inner Wall furtherincludes glycerin admixed to said collagen.

5. A vessel as set forth in claim 1, wherein said inner Wall furtherincludes an antibiotic admixed to said collagen.

References Cited UNITED STATES PATENTS 3,106,483 10/1963 Kline et al1l762.2 3,108,357 10/1963 Liebig 128-334 X 3,272,204- 10/1966 Artandi etal 128334 FOREIGN PATENTS 591,509 8/1947 Great Britain.

DALTON L. TRULUCK, Primal Examiner.

US. Cl. X.R.

